Antisense RNA-mediated reduction of p53 induces malignant phenotype in nontumorigenic rat urothelial cells.
p53 mutation is commonly associated with high-grade, high-stage human urothelial carcinomas. Recent studies suggest that p53 mutation in low-grade, low-stage bladder carcinomas may be correlated with the progression of the disease. In the present study, we used antisense RNA methodology in vitro to evaluate the significance of the loss of p53 function at an early stage of urinary bladder carcinogenesis. An immortalized nontumorigenic rat urothelial cell line (MYP3) that strongly expresses wild-type (WT) p53 was transfected with a plasmid (pcDL-SR alpha-296) containing a rat WT p53 cDNA in antisense orientation. The transfection resulted in a significant reduction in p53 mRNA expression and protein synthesis, in stimulation of anchorage-dependent growth, and in acquisition of anchorage-independent growth potential. Three such clones, when tested in athymic nude mice, all formed muscle-invasive, high-grade transitional cell carcinomas at s.c. injection sites. When cells were inoculated into an orthotopic site (urinary bladder), one of two antisense transfectants tested formed bulky tumors in the bladder in all seven nude mice and metastases to lungs in three of the seven mice. Analysis of these cells revealed a decrease in the expression of p21 (WAF1, sdi1, or CIP1) and retinoblastoma (Rb) gene product. Phosphorylation of Rb protein was not inhibited when the cells were starved. No significant difference was observed in the expression of p16 protein. In cell cycle analysis, all antisense transfectants tested escaped from G1 arrest by starvation. Furthermore, secretion of interleukin (IL)-6 into culture medium was increased significantly. Treatment with anti-IL-6 antibody suppressed anchorage-dependent growth. This study directly demonstrates that the loss of p53 function at an early stage of urothelial carcinogenesis may result in acquisition of a malignant phenotype by regulating IL-6 production as well as cell cycle related genes.
Okamoto
M
M
Department of Pathology, Northwestern University Medical School, Chicago, IL 60611, USA.
Hattori
K
K
Fujimoto
K
K
Tanaka
Y
Y
Gloosby
C L
CL
Oyasu
R
R
D000818:Animals; D002453:Cell Cycle; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D017931:DNA Primers; D016158:Genes, p53 / Q000187:drug effects; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000494:pharmacology*; D051381:Rats; D014162:Transfection; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology*; D019459:Urothelium / Q000187:drug effects / Q000473:pathology*
D017931:DNA Primers; D016372:RNA, Antisense; D016159:Tumor Suppressor Protein p53